The focus of these studies is on cell fate decisions in developing T cells. Signals through the TCR and other surface receptors determine whether thymocytes will survive, mature, or die. Precursor T cells undergo a testing process in the thymus to ensure that cells expressing useless or self-reactive TCRs do not mature. These selection processes (known as positive and negative selection) require TCR engagement with peptide-MHC ligands expressed by thymic stromal cells. Thymocytes also receive signals as they mature that direct them into specific lineages. Our studies and those of others indicate that the strength and/or duration of TCR signaling influence lineage choice. In addition, T cell fate may be determined by the integration of TCR signals with other developmental cues, such as those regulated by the Notch receptor. To examine the role of Notch in development, we generated Presenilin (PS) conditional mutant mice in which all Notch activity is prohibited in targeted tissues. With gene deletion in the thymus, the generation of CD4 SP thymocytes was inefficient with a diverse TCR repertoire, and severely compromised in mice expressing a single MHC2-restricted TCR. Diminished T cell production correlated with impaired TCR signaling in PS-deficient thymocyte precursors that could be rescued with an activated form of Notch. Although these loss-of function studies revealed that Notch functions in thymocyte positive selection, there was no evidence that Notch regulates CD4/CD8 T lineage commitment. [unreadable] [unreadable] While these results suggested a functional link between the TCR and Notch signaling pathways in thymocytes, it was unclear how these signaling pathways were related. To address how Notch regulation of target genes impacts thymocyte differentiation, we collaborated with Ellen Robeys lab to examine thymocyte subsets by gene expression profiling in Notch and PS mutants under different selecting conditions. Independently, we examined the effects of PS-deficiency on a broad range of genes. Despite the dramatic phenotype of PS-deficient precursor thymocytes, few genes were differentially expressed >2 fold. In agreement with our flow cytometric data, levels of CD5 were reduced, whereas mRNA levels of CD3, CD4, CD8, CD44, and CD69 were equivalent. No differences were observed in mRNA for proteins involved in proximal TCR signaling, e.g., Lck and ZAP70. Consistent with a failure to detect increased apoptosis in PS-deficient thymocytes, the expression for anti-apoptotic proteins, Bcl-2 and Bcl-XL, were normal. Since Notch often regulates the expression of transcription factors, one potentially intriguing difference was an increased expression of AP-1 family transcription factors in PS-deficient thymocytes. [unreadable] Obviously, analysis of mRNA would not reveal any changes in post-transcriptional or post-translational modifications, or changes in protein localization. To address these issues, we assayed TCR-stimulated PS-deficient thymocytes for total tyrosine-phoshorylation, or phosphorylated forms of ZAP70, Erk, Vav, Lck, GSK3, PTEN, PDK1, RAF, PLC-gamma1, Akt, and LAT (proteins mediating TCR signal transduction). The most interesting observation was an alteration in LAT, an adapter whose phosphorylation by ZAP70 leads to recruitment and activation of PLC-gamma1, PI3K, and Ras. LAT appears on gels as a doublet of p36 and p38 kD forms. While p36 predominates in control thymocytes, the p38 form was clearly increased relative to the p36 form in PS-deficient thymocytes. While yet to be established, work of others suggests that these two forms may reflect differences in palmitoylation and/or phosphorylation. Although our previous studies revealed defective Ca++ mobilization in response to TCR stimulation, this striking alteration in pLAT indicates that the effect of Notch on TCR signal transduction begins upstream of the Ca++ response in proximal TCR signaling events. Relevant to these findings, Ohashi et al. have reported that anergic T cells can be maintained in an unresponsive state via changes in palmitoylation of LAT, which regulates its recruitment to lipid rafts. [unreadable] [unreadable] Because we had uncovered a functional link between Notch and TCR signaling in thymocyte precursors undergoing positive selection, we were interested in whether there was a similar relationship between Notch and TCR signaling in nave CD4 T cells in response to antigen. Previous reports suggested that Notch signaling controls T helper (Th1/Th2) differentiation of nave CD4 T cells, although the mechanism responsible for this regulation was controversial. Analogous to the model for thymic development in which the quantity of TCR signal biases the CD4/CD8 T cell fate decision, there is a strength of signal model for Th1 /Th2 lineage commitment in mature T cell responses, where low doses of antigenic peptide promote Th2, and high doses of peptide promote Th1 polarization. To determine the role of Notch in T helper differentiation, we generated a new model in which conditional deletion of the PS1 gene begins late in thymocyte development and is completed in peripheral nave T cells. For collaborative experiments with William Pauls lab, we generated Presenilin-deficient H-2k RAGo 5C.C7 TCR transgenic mice to examine an antigen specific response. The number of nave/memory peripheral CD4 T cells in these mice was normal, consistent with the fact that there was no defect in thymic development. Previous studies of Notch mutants had used only a single antigen dose and analyzed a single late time point, making it impossible to distinguish between factors influencing early events in TH1/2 lineage commitment, from factors affecting maturation and/or maintenance of cytokine responses. In our experiments, purified naive CD4 T cells were stimulated with varying doses of specific peptide using splenic DC under non-polarizing conditions. At high doses of peptide, control T cells made predominantly an IFN-gamma response, but as peptide concentration was decreased, a gradual switch occurred, with less IFN-gamma and more IL-4, such that only IL-4 was produced at the lowest doses of peptide. In contrast, CD4 T cells of PS-deficient mice made only IL-4 and no IFNgamma at high doses of peptide and lowering the dose of peptide further reduced the IL-4 response, as well as cell division and recovery. Thus, at the highest doses of peptide, control CD4 T cells were polarized toward Th1, whereas PS-deficient CD4 T cells were strikingly polarized toward Th2. This phenotypic difference correlated with induction of GATA3 expression only in the mutant T cells. At low peptide concentration, GATA3 and IL-4 mRNA were equivalent in mutant and control cells at early time points, but IL-2 production was significantly reduced in mutant cells. Defects in IL-4 production at low doses of peptide could be rescued by addition of exogenous IL-2, suggesting IL-2 production to be the major defect early in the response. Interestingly, this altered pattern of GATA3, IL-2, and IL-4 expression in PS-deficient CD4 T cells is similar to results obtained with MAPK inhibition of stimulated control CD4 T cells. Rather than supporting a direct role for Notch in regulating Gata3 or IL-4 transcription as previously reported, these experiments with PS-deficient T cells suggest that the primary defect resulting from Notch inhibition is in TCR signaling. Thus, similar to developing thymocytes, there is a functional link between Notch and TCR signaling in T helper differentiation in peripheral T cells.